Inverse Solvent Isotope Effects in Enzyme-Catalyzed Reactions

Abstract

Solvent isotope effects have long been used as a mechanistic tool for determining enzyme mechanisms. Most commonly, macroscopic rate constants such as k cat and k cat /K m are found to decrease when the reaction is performed in D 2 O for a variety of reasons including the transfer of protons. Under certain circumstances, these constants are found to increase, in what is termed an inverse solvent kinetic isotope effect (SKIE), which can be a diagnostic mechanistic feature. Generally, these phenomena can be attributed to an inverse solvent equilibrium isotope effect on a rapid equilibrium preceding the rate-limiting step(s). This review surveys inverse SKIEs in enzyme-catalyzed reactions by assessing their underlying origins in common mechanistic themes. Case studies for each category are presented, and the mechanistic implications are put into context. It is hoped that readers may find the illustrative examples valuable in planning and interpreting solvent isotope effect experiments.

Dissecting the Mechanism of (R)-3-Hydroxybutyrate Dehydrogenase by Kinetic Isotope Effects, Protein Crystallography, and Computational Chemistry

“…For instance, if two or more proton transfers give rise to the solvent isotope effect but at least one of them produces a large enough inverse isotope effect, the overall observed isotope effect will be smaller than the largest individual normal one. 62 , 63 Surprisingly, D 2 O ( k cat / K M NADH ) = 2.0 ± 0.4, reflecting a decrease in C f NADH , similar to what was observed for the D ( k cat / K M NADH ) with 3-oxovalerate as substrate.…”

Controlling Non-Native B12 Reactivity and Catalysis in the Transcription Factor CarH

“…Quantitative kinetic analysis and proton inventory experiments with a suitable model system will be required to better clarify these possibilities. 31,33 Putting these findings together with the original mechanistic hypothesis of Gryko, 10 we propose that CarH*-catalyzed styrene alkylation proceeds via the mechanism shown in Figure 4. Catalysis is initiated by reduction of cobalamin to its Co(I) oxidation state (B), which undergoes oxidative addition with EDA to generate intermediate E. Addition of styrene to this complex, presumably via homolysis and recombination of organic radical and Co(II) intermediates, 17 would give a second Co(III)-alkyl complex.…”

Racemases and epimerases operating through a 1,1-proton transfer mechanism: reactivity, mechanism and inhibition

“…A second approach is to measure exchange of the a-proton with isotopically labelled substrates 82,95,[152][153][154] or solvent, 8,73,[78][79][80]107 measuring reaction extent by scintillation counting, mass spectrometry or NMR. Such approaches will introduce significant kinetic isotope effects [155][156][157][158] and deprotonation and reprotonation rates will be markedly different from each other although the extent of this will depend on levels of conversion of substrate and whether the transition states are early or late. 107 Consequently, careful design of experiments is needed, especially where precise rate measurements are required.…”